我们感谢迈克尔Reth，Schägger赫尔曼和玛格丽塔卡马乔的瓦哈尔科学支持。这项工作是由FORSYS从Bundesministerium附耳教化和Forschung（BMBF）的资助，由德意志研究联合会（DFG）的BIOSS，并在部分支持由德国联邦和各州政府的卓越倡议（GSC - 4，研究生院Spemann ）。

**本视频协议是基于相关出版物1：蓝色的原生聚丙烯酰胺凝胶电泳（BN - PAGE）多蛋白复合物的鉴定和分析。 Mahima斯瓦米，加布里埃尔M. Siegers，苏珊娜Minguet，贝恩德Wollscheid，和沃尔夫冈西澳Schamel。 STKE 2006年“科学”（345）：PL2，7月25日，2006年，[作者：10.1126/stke.3892006pl4] <a href="http://stke.sciencemag.org/cgi/content/abstract/sigtrans;2006/345/pl4">请点击这里查看本出版物</a> 。 1。透析细胞裂解液的制备<ol><li> 10 × 10 6细胞的收获和沉淀，离心350克5分钟，4 ° C 。 </li><li> 1 mL冰冷的PBS（配方1）液洗细胞沉淀三次，离心分离，在步骤1.1。 </li><li>悬浮颗粒和250μL冰冷的BN -裂解液（配方2）在冰上孵育15分钟。 </li><li> 13,000克离心15分钟4 ° C，移除不溶物。 </li><li>融化在第1.5 ml离心管使用巴斯德吸管加热大口径方孔，然后置于冰上管降温至4 ° C </li><li>步骤1.4转移到冷冻管与孔在第上清。 </li><li>放置一个与开管的顶部，接近上限钳透析膜（分子量10 kD的切断），并切断多余的透析膜伸出。 </li><li>一边仔细地用封口膜密封帽。 </li><li>反转管和离心机上下颠倒，持续10秒的最低速度，在50 mL锥形管在细胞培养离心机可能在4 ° C。卸下离心机使用镊子，以避免转向管右侧的倒管。 </li><li>准备一个100 mL烧杯中，用冷水BN透析液（配方3）和搅拌板。使用至少10毫升的BN透析每100μL样品缓冲。 </li><li>贴上管倒在烧杯内的磁带，使用弯曲的巴斯德吸管帽下方气泡从孔中删除。 </li><li>放在一块磁铁搅拌器顶部的烧杯中，开关上的搅拌器和放置6小时，或在寒冷的房间里过夜。偶尔检查，以确保搅拌创建气泡在透析膜。 </li><li>收集在一个新的冷冻离心管透析的细胞裂解液。 </li></ol> 2。浇注国民凝胶<ol><li>梯度凝胶浇注是在室温下进行的梯度混合器。因为聚丙烯酰胺是高度的神经毒性，必须戴手套。避免接触任何与SDS。 </li><li>广场上的轰动板梯度混合器，将它附加到了一块软管。使用的阀门关闭通道，并密切与管钳。到“高”缸油管相连，放置一个磁力搅拌器15％。 </li><li>主题成peristalitic泵软管，并附加一个注射器针头结束。然后，将针凝胶仪器的两个玻璃板之间。 </li><li>准备4％（配方5）和15％（配方6）分离胶解决方案，加入APS和TEMED前立即使用。合并后的卷应该是平等的分离胶量。 </li><li>倒入梯度混合器相应的气瓶（4％到“低”到“高”缸和15％），这些凝胶的解决方案。 </li><li>阀打开了通道连接在左缸用拇指按两个凝胶水库内的气泡和力量。 </li><li>磁力搅拌器上的开关，取出钳，peristalitic泵切换到5毫升每分钟。允许凝胶玻璃板之间缓缓流。确保针以上的液体总是。 </li><li>允许所有的液体进入凝胶的仪器，然后轻轻覆盖用异丙醇。允许凝胶聚合至少30分钟在室温。 </li><li>清洁浇筑设备立即与卫生署2 O（不使用清洁剂）。 </li><li>删除的异丙醇，洗净与卫生署2，删除用Whatman纸DH 2 O。 </li><li>准备了3.2％浓缩胶（配方），加入APS和TEMED前立即使用。 </li><li>倒入浓缩胶和分离胶上介绍玻璃板之间的梳子，避免气泡。浓缩胶聚合后，凝胶降温至4 ° C。 </li><li>样品装载前，立即取出梳子慢慢地，拉出来凝胶平面的角度。这使得空气进入的口袋迅速，从而提高了井的质量。 </li></ol> 3。 BN - PAGE分离细胞裂解液透析<ol><li> 1至40μL透析裂解液和10至20μL标记组合（配方10）负载在干井4 ° C。冷阴极的缓冲液（配方8），覆盖在每口井的样本。 </li><li>内腔填充冷猫HODE缓冲区，并用冷阳极缓冲外/下腔（配方）。 </li><li>应用100伏到minigel或150V一个大的凝胶，直到样品进入分离胶。运行凝胶在4 ° C。 </li><li>增加电压至180 V（minigel）或400V（大胶）和运行，直到染料前沿到达凝胶年底。运行需要3至4小时，为迷你，和18至24小时为一个大的凝胶。 </li></ol> 4。第二维的SDS - PAGE <ol><li> BN - PAGE的第一个维度里，一个定期为分子量标记线，并已透析裂解等分一个经常弄一个单一的大巷准备一个标准的10％SDS凝胶（食谱12-15） SDS样品缓冲液（配方11）混合，煮沸5分钟，在95 ° C使用垫片的厚度增加是由两层透明胶带，以简化的SDS凝胶上装载的BN - PAGE凝胶片。 </li><li>删除的BN - PAGE凝胶电泳仪板，轻轻地撬起来一个板块。 </li><li>取出浓缩胶和切出车道的BN - PAGE凝胶含有的蛋白质利益。 </li><li>将2X SDS样品缓冲液孵育的BN - PAGE凝胶片在室温为10分钟。 </li><li>煮的BN - PAGE凝胶片在微波炉中简要（不超过20秒）。 </li><li>再在室温下15分钟热SDS样品缓冲液中孵育的BN - PAGE凝胶片。 </li><li>负载的BN - PAGE凝胶片在大井，在浓缩胶的SDS - PAGE凝胶避免气泡，并覆盖SDS样品缓冲液中分得一杯羹。负载标记和裂解控制。 </li><li>根据标准协议执行电泳。 </li></ol> 5。 MPC的亚基免疫印迹检测<ol><li>转让，准备六的Whatman论文和PVDF膜装修SDS凝胶的大小。 </li><li>在30秒的100％甲醇孵育PVDF膜和转移的缓冲液（配方16）中浸泡的Whatman论文。 </li><li>将三个文件的Whatman，聚偏氟乙烯膜，SDS凝胶（删除浓缩胶），并再而三的Whatman在成半干转移细胞的结构类似于三明治的论文。 </li><li>适用于20 V 25分钟。 </li><li>检测蛋白质按标准免疫协议。 </li></ol> 6。代表性的成果我们目前的货币政策委员会表征真核生物19S，20S和26S蛋白酶体作为一个例子分析2D BN / SDS - PAGE电泳（图1A）。 HEK293细胞裂解用缓冲液含0.1％的Triton X - 100的洗涤剂破坏的膜和溶解膜蛋白复合物。这些细胞裂解液对BN透析缓冲液透析去除盐和小代谢产物。然后由第二维的SDS - PAGE梯度4-15％的BN - PAGE分离，储值卡。蛋白与抗β2亚基和20S蛋白酶体Mcp21抗体免疫印迹可视化。 <img src="/files/ftp_upload/2164/2164fig1.jpg" alt="图1" /> 图1。一个两维BN-PAGE/SDS-PAGE的方法，用细胞裂解液（一）从细胞裂解液的2D BN-PAGE/SDS-PAGE方法的流程图。 （二）一个二维BN-PAGE/SDS-PAGE结构的计划。天然条件下在第一维BN - PAGE分离蛋白质和储值卡。对于第二个层面，蛋白质和/或储值卡，经SDS变性凝胶地带的BN - PAGE分离后，随后的SDS - PAGE。单体的蛋白质会在双曲对角线由于在第一梯度凝胶电泳和第二维线性凝胶迁移。一个具体的货币政策委员会的组件将被发现的对角线，低于位于一条垂直线。 它已被证明，第一维BN和第二维的SDS - PAGE的组合，在双曲对角线由于梯度凝胶的单体蛋白质在第一和第二个维度（线性凝胶迁移（卡马乔瓦哈尔， Wollscheid等等2004）;图1B）。多用途储值卡的组件都位于低于此对角线。代表相同的货币政策委员会亚基的蛋白质，可以发现一个在第二个维度垂直线，而相同的蛋白质在一个水平线上的几个点表明，在几个不同的储值卡中的蛋白质的存在。图2显示，在我们的实验β2和Mcp21免疫印迹显示特定的蛋白质复合物含有这些蛋白酶体亚基的存在。这两种蛋白质检测安排在一个水平线上的个别景点，这表明，β2和Mcp21代表选民的几个不同的储值卡。这些储值卡可以清楚确定的26S蛋白酶体（20S加19S第），20S蛋白酶体调节亚基PA28一起，20S蛋白酶体单独的规模和组成的基础上，。两者合计，这些热塑LTS表明，内源性的多用途储值卡可以由一个二维BN-PAGE/SDS-PAGE使用细胞裂解液的方法识别和特点。此方法适用于多用途储值卡的大小，组成和相对丰度的决心。 <img src="/files/ftp_upload/2164/2164fig2.jpg" alt="图2" /> 图2。免疫后两维BN-PAGE/SDS-PAGE真核蛋白酶体识别和分析真核蛋白酶。不同形式的检测，0.1％的Triton X - 100的HEK293细胞裂解。细胞裂解液，透析，随后遭受的BN - PAGE（4-15％），单独的储值卡。第二维的SDS - PAGE（10％）之后，运行规模的个别子的分离。免疫是承认Mcp21和β2亚基20S核心复杂和调节亚基PA28的特异性抗体。 一表的特定试剂（按字母顺序排列）： <table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 评论 </td></tr><tr><td> 6 - 氨基己酸 （ε-氨基己酸） </td><td> Sigma - Aldrich公司，德国Taufkirchen的， </td><td>这种化学物质是一种刺激性，应带手套处理。 </td></tr><tr><td>丙烯酰胺，双丙烯酰胺溶液（40％），混合32:1 </td><td> Applichem，德国达姆施塔特</td><td>这个解决方案是神经毒性，应带手套处理。 </td></tr><tr><td>双三</td><td>罗斯，卡尔斯鲁厄，Germa - NY </td><td></td></tr><tr><td> Brij 96 </td><td> Sigma - Aldrich公司，德国Taufkirchen的， </td><td></td></tr><tr><td>考马斯亮蓝G250 </td><td>赛瓦，海德堡，德国</td><td>不要代替其他类型的考马斯如考马斯亮蓝R250或胶体COO - Massie的蓝色染料。 </td></tr><tr><td>毛地黄皂苷</td><td> Sigma - Aldrich公司，德国Taufkirchen的， </td><td>毛地黄皂苷是有毒的。处理缓冲区或样品含有这种威慑的绅士时，必须戴上手套。 </td></tr><tr><td> Dodecylmaltoside </td><td> Applichem，德国达姆施塔特</td><td></td></tr><tr><td> TRITON X - 100 </td><td>罗斯，卡尔斯鲁厄，Germa - NY </td><td> TRITON X - 100是有毒的。处理缓冲区或样品含有这种洗涤剂时，必须戴上手套。 </td></tr></tbody></table>二。具体的材料和设备表： <table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td></tr><tr><td>透析膜（截止10日至50 kD的分子量） </td><td>罗斯，卡尔斯鲁厄，德国</td></tr><tr><td>凝胶电泳系统</td><td>例如，来自Bio - Rad，德国慕尼黑</td></tr><tr><td>梯度混合器</td><td>自制或商用Bio - Rad公司，德国慕尼黑</td></tr><tr><td>蠕动泵</td><td> Amersham公司Pharmacia公司生物技术，德国弗赖堡</td></tr><tr><td>聚偏氟乙烯（PVDF）膜</td><td> Millipore公司，位于Eschborn，德国的Immobilon - P </td></tr><tr><td>半干转移设备</td><td>例如，来自Bio - Rad，德国慕尼黑</td></tr><tr><td>硅管（直径3至5毫米，长1米） </td><td> NeoLab，德国海德堡</td></tr></tbody></table>三。配方的表： <table border="1"><tbody><tr><td> 号 </td><td> 缓冲器和解决方案 </td><td> 内容 </td><td> 评论 </td></tr><tr><td> 1 </td><td>磷酸盐缓冲液（PBS） </td><td> NA 2 HPO 4 8.1 mMKH 2 PO 4 1.5毫米 氯化钠138毫米 氯化钾2.7毫米</td><td>如果前比较正确的解决方案应该是pH值7.4。 </td></tr><tr><td> 2 </td><td> BN -裂解液</td><td> 相应的缓冲区 二 - 三20毫米 ε-氨基己酸500毫米 氯化钠20毫米 EDTA，pH值8.0 2毫米 甘油10％ 用HCl调节pH至7.0。保存在4 ° C。 洗涤剂 毛地黄皂苷0.5至1.0％ 或Brij 96 0.1至0.5％ 或Triton X - 100 0.1〜0.5％ 或Dodecylmaltoside 0.1％至0.5％ 蛋白酶和磷酸酶抑制剂 抑肽酶10微克/毫升 亮肽素10微克/毫升 PMSF 1毫米 氟化钠0.5毫米 钒酸钠0.5毫米</td><td>适当的清洁剂，必须凭经验确定，并应在其他裂解液配方中使用的相同。 毛地黄皂苷必须加上之前从DH 2 O（存储在5毫升分装在-20 ° C）的2％原液使用。 使用前应加入蛋白酶和phophatase抑制，口服补液盐。 当钠orthova nadate此外，该缓冲区会变得颜色偏黄。 </td></tr><tr><td> 3 </td><td> BN透析缓冲器</td><td> 相应的缓冲区 二 - 三20毫米 ε-氨基己酸500毫米 氯化钠20毫米 EDTA，pH值8.0 2毫米 甘油10％ 用HCl调节pH至7.0。保存在4 ° C。 洗涤剂 毛地黄皂苷0.3％至0.5％ 或Triton X - 100 0.1％ 或Brij 96 0.1％ 或Dodecylmaltoside 0.1％ 蛋白酶和磷酸酶抑制剂 PMSF 1毫米 钒酸钠0.5毫米</td><td>适当的清洁剂，必须凭经验确定，并应在其他裂解缓冲液中使用的相同，但表示在低浓度的。 洗涤剂必须添加到预泄聚集在凝胶电泳的堆叠步骤。 使用前应加入蛋白酶和phophatase抑制，口服补液盐。 </td></tr><tr><td> 4 </td><td> 3倍的BN -胶缓冲液</td><td>双 - 三150毫米 ε-氨基己酸200毫米 用HCl调节pH至7.0。保存在4 ° C。 </td><td></td></tr><tr><td> 5 </td><td> 4％，分离胶</td><td> 3倍的BN -胶缓冲液（配方4）5.00毫升 丙烯酰胺/双丙烯酰胺1.50毫升 生署2 O 8.50毫升 黄芪多糖，在卫生署2 O 54μL的10％ TEMED 5.4微升</td><td>加入APS和TEMED immedia - tely浇筑前凝胶，这些试剂促进聚合。 这食谱足以投出30毫升的凝胶。调整卷的数量和大小的凝胶被浇。 </td></tr><tr><td> 6 </td><td> 15％的分离胶</td><td> 3倍的BN -胶缓冲液（配方4）5.00毫升 丙烯酰胺/双丙烯酰胺5.63毫升 甘油70％，4.38毫升 黄芪多糖，在卫生署2 O 42μL的10％ TEMED 4.2微升</td><td>加入APS和TEMED immedia - tely浇筑前凝胶，这些试剂促进聚合。 这食谱足以投出30毫升的凝胶。调整卷的数量和大小的凝胶被浇。 丙烯酰胺，双丙烯酰胺的浓度也可以多种多样，从10％至18％的必要。 </td></tr><tr><td> 7 </td><td> 3.2％的浓缩胶</td><td> 3倍的BN -胶缓冲液（配方4）3.00毫升 丙烯酰胺/双丙烯酰胺0.72毫升 生署2 O 5.28毫升 黄芪多糖，在卫生署2 120μL，10％ TEMED 12微升</td><td>加入APS和TEMED immedia - tely浇筑前凝胶，这些试剂促进聚合。 这食谱足以投出30毫升的凝胶。调整卷的数量和大小的凝胶被浇。 </td></tr><tr><td> 8 </td><td>阴极缓冲</td><td>二 - 三15毫米 甘氨酸50毫米 考马斯亮蓝G250 0.02％ 准备1公升为10倍的股票，调整pH至7.0，用HCl，并储存在4 ° C。 使用前，应与卫生署2稀释的1:10。 </td><td>不要代替其他类型考马斯染料，如考马斯亮蓝R250或胶体考​​马斯亮蓝调。 </td></tr><tr><td> 9 </td><td>阳极缓冲</td><td>二 - 三50毫米 准备1公升为10倍的股票，调整pH至7.0，用HCl，并储存在4 ° C。 使用前，应与卫生署2稀释的1:10。 </td><td></td></tr><tr><td> 10 </td><td>标记混合</td><td>醛缩酶（158 KD）10毫克/毫升 过氧化氢酶（232 KD）10毫克/毫升 铁蛋白（440和880 KD）10毫克/毫升 甲状腺球蛋白（670 KD）10毫克/毫升 BSA（66和132 KD）10毫克/毫升 二 - 三20毫米 氯化钠20毫米 甘油10％ 用HCl调节pH至7.0。保存在4 ° C。 </td><td>分子量标记也从几个来源，其中包括的体外根或法玛西亚市售。 </td></tr><tr><td> 11 </td><td> SDS样品缓冲液</td><td>三12.5毫米 SDS的4％ 甘油20％ 溴酚蓝0.02％ 调节pH值至6.8。为了减少二硫键，加9毫升β-巯基乙醇。 </td><td>作为一个粉和β- MER - captoethanol SDS是有毒的。因此，使用手套和引擎盖下工作。 </td></tr><tr><td> 12 </td><td> 4X降低缓冲区</td><td>三1.5米 SDS的0.4％ 调节pH值至8.8。 </td><td> SDS的粉末是有毒的。因此，使用手套和引擎盖下工作。 </td></tr><tr><td> 13 </td><td> 4X上层缓冲区</td><td>三0.5 M SDS的0.4％ 调节pH值至6.8。 </td><td> SDS的粉末是有毒的。因此，使用手套和引擎盖下工作。 </td></tr><tr><td> 14 </td><td> 10％分离胶</td><td>丙烯酰胺（30％）2.0毫升 4X较低的缓冲液1.5毫升 生署2 O 2.454毫升 黄芪多糖，在卫生署2 O 40μL的10 ％ TEMED 6微升</td><td>加入APS和TEMED immedia - tely浇筑前凝胶，这些试剂促进聚合。 这食谱足以投出30毫升的凝胶。调整卷的数量和大小的凝胶被浇。 </td></tr><tr><td> 15 </td><td> 4.8％的浓缩胶</td><td>酰胺（30％）320μL， 4X上层缓冲液500μL 生署2 O 1.16毫升 APS的，卫生署2的10％：20 μL TEMED 2微升</td><td>加入APS和TEMED immedia - tely浇筑前凝胶，这些试剂促进聚合。 这食谱足以投出30毫升的凝胶。调整卷的数量和大小的凝胶被浇。 </td></tr><tr><td> 16 </td><td>半干转移缓冲器</td><td>三48毫米 甘氨酸39毫米 甲醇20％ SDS的0.1％ 音量调整到1升，与DH 2 O在室温下储存。 </td><td>粉末和甲醇SDS是有毒的。因此，使用手套和引擎盖下工作。 </td></tr></tbody></table>

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	<li>Arnold, I., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+F1F0-ATP+synthase+exists+as+a+dimer:+identification+of+three+dimer-specific+subunits.">Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.</a> EMBO J. 17 (24), 7170-7178 (1998).</li><li>Camacho-Carvajal, M. M., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol. Cell. 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W., Reth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+and+oligomeric+complexes+of+the+B+cell+antigen+receptor.">Monomeric and oligomeric complexes of the B cell antigen receptor.</a> Immunity. 13 (1), 5-14 (2000).</li><li>Swamy, M., Minguet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+native+antibody-based+mobility-shift+technique+(NAMOS-assay)+to+determine+the+stoichiometry+of+multiprotein+complexes.">A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.</a> J Immunol Methods. 324 (1-2), 74-83 (2007).</li><li>Swamy, M., Siegers, G. 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在这项研究中，我们描述的多用途储值卡的BN - PAGE分析。二维的方法是使用天然条件下的第一次单独的储值卡，然后进一步细分到其各个组成部分，由第二维的SDS - PAGE。 样品制备细胞裂解液。对于许多的多用途储值卡的增溶，适当的清洁剂是必要的，保留的蛋白质复合物的结构。在这里，我们使用0.1％的Triton X - 100的。然而，最佳的清洁剂和适宜浓度必须确定每MPC经验。的Triton X - 100的情况下，例如，它已经报道，洗涤剂浓度低允许确定一个二聚体形式的F 1 F 0 - ATP酶复合物（阿诺德， 普发等 ，1998 ）。高等教育TRITON X - 100的浓度，然而，导致二聚体解离的单体 F 1 F 0 - ATP酶复合体的相应增加。这与我们以前的研究之一，我们表明，多价，T细胞受体复合物（TCR）的提取与Brij 96的低浓度时保留，而浓度较高，或另一种洗涤剂的使用所谓的毛地黄皂苷结果提取单体的TCR（Schamel，Arechaga 等，2005）。常用的洗涤剂，可以测试包括毛地黄皂苷（0.5％至1％），TRITON X - 100（0.1至0.5％），Brij 96（0.1％至0.5％），或dodecylmaltoside（0.1％至0.5％）。这些试剂均为非离子型洗涤剂，这往往是最适合MPC稳定。请注意，应避免接触，与SDS和其他强洗涤剂（卡马乔的瓦哈尔，Wollscheid等，2004） 。 透析需要的细胞裂解液，以实现货币政策委员会在BN -凝胶分离（卡马乔瓦哈尔，Wollscheid 等2004）;（海斯，Junkes 等 2005）。看来，盐的浓度或去除低分子量杂质的调整是高分辨率的关键。值得注意的是，膜制剂和储值卡，已immunopurified后来就从抗体洗脱，适合BN - PAGE（斯瓦米，Siegers 等，2006）。在这两种情况下，样品没有要的BN - PAGE分离，如果膜溶解或洗脱的BN -裂解液进行透析。 BN - PAGE对蛋白质的分离，染料考马斯亮蓝需要，结合unspecifically蛋白质和覆盖带负电荷。从而，考马斯蓝可在中性pH（Schägger和1991年冯Jagow）的蛋白质对阴极电泳迁移;（Cramer等Schägger，1994年）。此外，考马斯蓝阻止蛋白质聚集在电泳过程中的浓缩胶。 BN - PAGE，考马斯亮蓝G250考马斯亮蓝R250或胶体考​​马斯亮蓝调，而不是要使用。 在运行BN - 凝胶之前，它是必要的，以确保适合凝胶的百分比利益MPC的预期大小。预制与不同梯度和适当的缓冲区是自Invitrogen（NativePAGE NOVEX双三凝胶体系）市售的BN -凝胶。但英国国民凝胶也可以使用与persistaltic泵的梯度混合器准备。为了保证一个完整的渐变，液体应在浇筑，并应避免气泡流不断。我们建议加载到不同的凝胶样品稀释，因为超载可能导致在电泳过程中蛋白质沉淀。此外，应运行的BN -凝胶，在4 ° C到防止蛋白质的降解，并保持储值卡完好。 BN - PAGE，储值卡的可视化后，可以通过考马斯亮蓝染色，银染或免疫。考马斯亮或银染色可视化蛋白条带，适合作进一步的分析，通过质谱（卡马乔的瓦哈尔，Wollscheid等， 2004）。在免疫的情况下，感兴趣的多用途储值卡的最佳传输条件要凭经验确定。要知道，考马斯蓝也是在一个BN - 凝胶印迹转移。因此，凝胶将无色的成功转让后，而膜将对蓝色。此外，重要的是要提，不是每一个主要的抗体，用于SDS - PAGE后检测工作，是适用于根据BN - PAGE免疫印迹。它可以发生抗体不承认利益MPC的，因为他们的抗原表位是隐藏的蛋白质天然构象。为了克服这个问题，很可能在转让之前，由沸腾不久在1X SDS样品缓冲液凝胶BN - 凝胶内的蛋白质变性。 在我们的例子中，我们并没有受到直接的BN - 凝胶检测蛋白条带。相反，我们进一步除以第二dimensi BN - PAGE分离裂解在SDS - PAGE。在第二维SDS凝胶，单体蛋白在双曲对角线由于在第一和第二个维度（卡马乔的瓦哈尔，Wollscheid 等，2004）的线性凝胶梯度凝胶迁移。这使得易于识别的储值卡，因为它们是低于这个双曲线​​对角线本地化。子组件的一个独特的货币政策委员会在第二维的SDS - PAGE垂直线分开。组件的几个dinstinct储值卡的选民可以在一个水平线上，根据MPC的大小确定。但是，它必须考虑到多个蛋白斑点出现在一个垂直线，也可以单独的复合物，迁移在同一位置的BN - PAGE的一部分。最后证明，他们是在相同的货币政策委员会目前可以通过抗体为基础的凝胶迁移实验。在这个实验中，细胞裂解液是一种抗体孵育，反对由一个确定的点之前的BN - PAGE表示的一种蛋白质。这样的结果在所有的多用途储值卡包含对​​这种蛋白的分子质量较高的，在第一维转变。其他蛋白质，这些储值卡的一部分，将经过这个复杂的具体的转变，因此容易识别，在第二维SDS凝胶。 组成的多用途储值卡不仅可以BN - PAGE分析，但也可能确定其化学计量（2000年Schamel和Reth）（Schamel 2001年），（斯瓦米，Minguet等2007 ）。为此，NAMOS法（以抗体为基础的的移动移法）可以执行。在抗体为基础的的凝胶迁移实验，细胞裂解液培养单克隆亚基特异性抗体。这将导致在诱导国民凝胶电泳immunoshifts，允许从推理移位的程度，对多用途储值卡的化学计量 conlusion，BN - PAGE是适合多用途储值卡的识别和他们的规模，构成，以及相对丰度的决心。作为NAMOS检测执行的，它也提供了可能性，确定一定MPC的化学计量。由于其普遍适用性，这项技术是一个非常有用的工具为表征（德克尔，Muller 等 1996年）的多用途储值卡;（2008年维蒂希和Schagger）;（瓦格纳，Rehling等2009）;（2009年维蒂希和Schägger） 。

blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates

多蛋白复合物（储值卡）起到至关重要的作用，在与其他蛋白质的功能或监管的复合物（贝，格兰泽等 ，2003）发现，因为大多数的蛋白质可以在细胞信号。因此，研究蛋白质相互作用网络需要细致刻画的多用途储值卡获得的蛋白质功能和调节的综合理解。对于识别和分析，储值卡必须分开天然条件下。在这个视频中，我们描述的多用途储值卡的蓝色原生聚丙烯酰胺凝胶电泳（BN - PAGE）的分析。 BN - PAGE是一种技术，使具有较高的分辨率比凝胶过滤或蔗糖密度离心提供的天然构象的储值卡分离，因此可用来确定货币政策委员会的规模，组成和相对丰度（Schägger和冯Jagow 1991年） （Cramer 等 Schägger，1994年）。通过这种方法，根据流体力学尺寸和形状在聚丙烯酰胺基质蛋白分开。在这里，我们展示的多用途储值卡的总细胞裂解液的分析，指出裂解液透析是关键的一步，使这些生物样本的BN - PAGE适用。使用第一个维度BN和第二维的SDS - PAGE的组合，我们的BN - PAGE分离储值卡可以进一步细分成各成分的经SDS - PAGE。凝胶分离后的MPC组件的可视化是执行标准的免疫印迹。作为货币政策委员会的BN - PAGE分析的例子，我们选择了良好的特点真核生物19S，20S和26S蛋白酶体。

Watch this Scientific Journal Video about Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates at JoVE.com

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

在这个视频中，我们描述的多蛋白复合物（储值卡）蓝色原生聚丙烯酰胺凝胶电泳（BN - PAGE）的表征。在第一个层面，透析细胞裂解液的BN - PAGE分离，以确定个人的多用途储值卡。在第二维SDS - PAGE电泳，利益的储值卡是进一步细分免疫印迹分析其成分。

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

130.3K 次观看.  University of Freiburg. 在这个视频中，我们描述的多蛋白复合物（储值卡）蓝色原生聚丙烯酰胺凝胶电泳（BN - PAGE）的表征。在第一个层面，透析细胞裂解液的BN - PAGE分离，以确定个人的多用途储值卡。在第二维SDS - PAGE电泳，利益的储值卡是进一步细分免疫印迹分析其成分。

视频: Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

变性梯度凝胶电泳（DGGE）

科研

生物学

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

粘蛋白琼脂糖凝胶电泳：蛋白质印迹高分子量糖蛋白


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

蓝色原代聚丙烯酰胺凝胶电泳免疫印迹法诱导人细胞线粒体呼吸链配合物的分析

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...